Tissue culture method of bulbus fritillariae cirrhosae

Abstract

The invention relates to a bulbus fritillariae cirrhosae tissue culture method, and relates to the technical field of bulbus fritillariae cirrhosae tissue culture, the bulbus fritillariae cirrhosae tissue culture method comprises the following steps: selecting a fresh and healthy bulbus fritillariae cirrhosae bulb as an explant, and then successively carrying out pretreatment before inoculation, pre-culture, induction culture, subculture, multiplication culture and rooting culture; the pretreatment before inoculation comprises the following steps: performing low-temperature plasma treatment on bulbus fritillariae cirrhosae bulbs, then cutting the bulbus fritillariae cirrhosae bulbs, soaking the bulbus fritillariae cirrhosae bulbs in an aqueous solution containing polyvinylpyrrolidone and carboxymethyl chitosan, and the like. On the basis of an existing bulbus fritillariae cirrhosae tissue culture method, the invention provides a novel bulbus fritillariae cirrhosae tissue culture method based on low-temperature plasma pretreatment and cis-3-hexene-1-alcohol chemical induction, the browning phenomenon in the culture process can be effectively inhibited, the culture efficiency is improved, and the bulbus fritillariae cirrhosae tissue culture method has the characteristics of low browning rate, high induction rate and the like; and a new means is provided for rapid large-scale propagation of the bulbus fritillariae cirrhosae.

Description

Technical Field

[0001] The present invention relates to the technical field of tissue culture of Fritillaria cirrhosa, and particularly relates to a tissue culture method of Fritillaria cirrhosa. Background Art

[0002] As a plant with extremely high medicinal value in the field of traditional Chinese medicine, Fritillaria cirrhosa has a long application history. Its dried bulbs contain rich bioactive components such as alkaloids, saponins, and polysaccharides. These active components endow Fritillaria cirrhosa with significant pharmacological effects such as clearing heat and moistening the lungs, resolving phlegm and relieving cough, and dispersing nodules and dissipating swelling. In clinical practice, Fritillaria cirrhosa is widely used in the treatment of various diseases such as dry cough due to lung heat, dry cough with little phlegm, consumptive cough due to yin deficiency, blood streaks in phlegm, scrofula, breast abscess, and lung abscess. It is a key raw material for many traditional Chinese medicine prescriptions and patent medicines (such as Fritillaria cirrhosa and loquat cough syrup, snake bile and Fritillaria cirrhosa liquid, etc.), and occupies an important position in the traditional Chinese medicine system.

[0003] However, the natural reproduction ability of Fritillaria cirrhosa is relatively weak, facing many reproduction problems. Its seed germination rate is low, the growth cycle is long, and it has strict requirements for the growth environment. These factors have led to a decreasing wild Fritillaria cirrhosa resource, making it difficult to meet the increasing market demand, and posing a severe challenge to the sustainable supply of Fritillaria cirrhosa. Therefore, exploring an efficient and reliable method to achieve the rapid reproduction of Fritillaria cirrhosa and the preservation of excellent varieties has become a key problem to be solved urgently.

[0004] As an effective means of rapid propagation and preservation of excellent varieties, tissue culture technology plays a crucial role in the large-scale production of Fritillaria cirrhosa. Currently, the tissue culture method of Fritillaria cirrhosa mainly includes the following key steps: 1. Selection and disinfection of explants: Carefully select healthy and pest-free Fritillaria cirrhosa bulbs as explants, and use chemical disinfectants (such as mercuric chloride, sodium hypochlorite, etc.) to perform surface disinfection treatment on them to minimize microbial contamination and create good aseptic conditions for the subsequent culture process.

[0005] 2. Pre-culture: Place the disinfected explants on a sterile medium for short-term pre-culture. This step aims to promote the explants to gradually adapt to the culture environment and prepare for the subsequent induction culture.

[0006] 3. Induction culture: By precisely adjusting the hormone ratio in the medium (such as the ratio of auxin to cytokinin), induce the explants to form callus or directly differentiate adventitious buds. This is a relatively crucial link in the tissue culture process and can affect the subsequent propagation effect.

[0007] 4. Subculture: After dividing the induced callus or adventitious buds, transfer them to a new medium for continuous culture to expand the propagation coefficient and achieve the rapid proliferation of Fritillaria cirrhosa.

[0008] 5. Proliferation culture: By further optimizing the culture conditions, such as light, temperature, nutrient components, etc., further improve the proliferation rate of callus or adventitious buds and improve the propagation efficiency.

[0009] 6. Rooting culture: Transfer the proliferated shoots without roots to a rooting medium to promote root growth, and finally form complete plants to complete the entire tissue culture process.

[0010] Although certain progress has been made in the tissue culture of Fritillaria cirrhosa D. Don in the prior art, such as CN110100730A, CN103621406A, CN112970589A, CN114847159A, etc., there are still many deficiencies. Among them, explant browning is a common and serious problem that affects the culture effect. The browning phenomenon not only hinders the normal growth and differentiation of explants, but also significantly reduces the induction rate and propagation coefficient, and even leads to the failure of the entire culture process. The main reason for explant browning is that phenolic substances are oxidized into quinone substances under the catalysis of polyphenol oxidase, and these quinone substances further polymerize to form brown substances, resulting in explant browning. In addition, although traditional chemical disinfection methods can effectively kill microorganisms, they often cause a certain degree of damage to explants, thereby affecting their growth and differentiation ability and resulting in poor culture effects. Moreover, the existing technology has a long culture cycle, cannot meet the needs of large-scale production, and limits the industrial development of Fritillaria cirrhosa D. Don.

[0011] Therefore, in view of the deficiencies of the existing technology, it is of great practical significance and application value to develop a new tissue culture method for Fritillaria cirrhosa D. Don. Summary of the Invention

[0012] To solve the above problems, the present invention provides a tissue culture method for Fritillaria cirrhosa D. Don.

[0013] The present invention provides a tissue culture method for Fritillaria cirrhosa D. Don, and the tissue culture method for Fritillaria cirrhosa D. Don includes: selecting fresh and healthy Fritillaria cirrhosa D. Don bulbs as explants, and then successively performing pre-inoculation pretreatment, pre-culture, induction culture, subculture, proliferation culture and rooting culture; The pre-inoculation pretreatment includes the following process: subjecting the Fritillaria cirrhosa D. Don bulbs to low-temperature plasma treatment, then cutting and soaking them in an aqueous solution containing polyvinylpyrrolidone and carboxymethyl chitosan; The working condition parameters of the low-temperature plasma treatment include: using a mixed gas composed of argon and oxygen with a flow rate of 100-150 ml / min as the working medium, a discharge power of 20-30 W, and a treatment time of 30-50 seconds; Both the medium used in the pre-culture and the induction culture contain cis-3-hexen-1-ol.

[0014] Further, the volume ratio of the argon gas to the oxygen gas is (5-8):1, the discharge power is 26 W, and the treatment time is 40 seconds.

[0015] Further, the concentration of the polyvinylpyrrolidone is 1.5-2.5 g / L.

[0016] Further, the concentration of the carboxymethyl chitosan is 3-5 g / L.

[0017] Further, the working condition parameters of the pre-culture include: Inoculated in the following medium: MS with a pH of 5.5-6.0 + 25-35 g / L sucrose + 5.5-6.5 g / L agar + 0.5-1.0 mg / L 6-BA + 0.8-1.5 mg / L NAA + 0.2-0.6 mg / L 2,4-D + 10-14 mg / L cis-3-hexen-1-ol; The culture conditions are as follows: temperature 21-23°C, relative air humidity 70-80%, light culture for 7-10 days, light time 12 hours / day, and light intensity 700-900 Lux.

[0018] Further, the working condition parameters of the induction culture include: Inoculated in the following medium: MS with a pH of 5.5-6.0 + 25-35 g / L sucrose + 6.5-7.0 g / L agar + 0.5-1.0 mg / L IBA + 0.8-1.5 mg / L NAA + 0.4-0.8 mg / L 2,4-D + 15-18 mg / L cis-3-hexen-1-ol; The culture conditions are as follows: temperature 21-23°C, relative air humidity 70-80%, light time 12 hours / day, light intensity 1950-2050 Lux, and culture time 12-15 days.

[0019] Further, the working condition parameters of the subculture include: Inoculated in the following medium: MS with a pH of 5.5-6.0 + 25-35 g / L sucrose + 6.5-7.0 g / L agar + 0.5-1.0 mg / L IBA + 1.0-1.5 mg / L KT + 0.1-0.3 g / L activated carbon; The culture conditions are as follows: temperature 21-23°C, relative air humidity 70-80%, light time 12 hours / day, light intensity 1950-2050 Lux, and culture time 14-17 days.

[0020] Further, the working condition parameters of the proliferation culture include: Inoculated on the following medium: MS with a pH of 5.5 - 6.0 + 25 - 35 g / L sucrose + 6.5 - 7.0 g / L agar + 0.5 - 1.0 mg / L IBA + 0.8 - 1.5 mg / L NAA + 0.5 - 1.0 mg / L 2,4 - D; Cultured at a temperature of 21 - 23°C, relative air humidity of 70 - 80%, light duration of 12 hours per day, light intensity of 1600 - 1800 Lux, and the culture time is 15 - 18 days.

[0021] Furthermore, the working condition parameters of the rooting culture include: Inoculated on the following medium: MS with a pH of 5.5 - 6.0 + 25 - 35 g / L sucrose + 6.5 - 7.0 g / L agar + 0.5 - 1.0 mg / L IBA + 1.5 - 2.0 mg / L KT + 1.5 - 2.0 mg / L NAA; Cultured at a temperature of 21 - 23°C, relative air humidity of 70 - 80%, light duration of 12 hours per day, light intensity of 2400 - 2600 Lux, and when the Fritillaria cirrhosa small bulbs grow to 5 - 6 cm in height and 5 - 10 roots grow out, hardening-off and transplanting are carried out.

[0022] The above technical solutions provided by the embodiments of the present invention have at least the following advantages compared with the prior art: The embodiments of the present invention provide a tissue culture method for Fritillaria cirrhosa. On the basis of the existing tissue culture method for Fritillaria cirrhosa, the present invention proposes a new tissue culture method for Fritillaria cirrhosa based on the synergistic effect of low-temperature plasma pretreatment and cis-3-hexen-1-ol chemical induction, which can effectively inhibit the browning phenomenon during the culture process, improve the culture efficiency, and has the characteristics of low browning rate and high induction rate, providing a new means for the rapid large-scale propagation of Fritillaria cirrhosa. Specifically: On the one hand, the present invention controls key parameters such as discharge power and treatment time by performing low-temperature plasma treatment on the Fritillaria cirrhosa bulb explants before inoculation and culture, achieving efficient sterilization and activity excitation of the Fritillaria cirrhosa bulb explants. At the same time, it can also avoid damage to the Fritillaria cirrhosa bulb explants caused by too high discharge power or too long treatment time and the toxic damage of traditional chemical disinfection reagents containing mercury and other substances to plant tissue cells, which is beneficial to promoting the formation of subsequent callus, reducing the overflow of phenolic substances, thereby inhibiting browning and increasing the induction rate.

[0023] On the other hand, the present invention discovers that cis-3-hexen-1-ol can significantly inhibit the activity of polyphenol oxidase and further applies it to the tissue culture of Fritillaria cirrhosa. By inhibiting the activity of polyphenol oxidase, the browning of tissues during the culture process is curbed, thereby further reducing the browning rate and increasing the induction rate. Specific embodiments

[0024] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the scope of protection of the present invention.

[0025] Unless otherwise specifically stated, all kinds of raw materials, reagents, instruments, and equipment used in the present invention can be obtained through market purchases or prepared by existing methods. For example, in the following examples and comparative examples, polyvinylpyrrolidone (CAS No.: 9003-39-8), carboxymethyl chitosan (CAS No.: 83512-85-0), cis-3-hexen-1-ol (CAS No.: 928-96-1), MS medium, 6-BA (6-benzylaminopurine), NAA (naphthaleneacetic acid), 2,4-D (2,4-dichlorophenoxyacetic acid), IBA (indolebutyric acid), KT (kinetin), etc. can all be commercially available products; at the same time, for the steps and parameters involved, without special limitations or specific descriptions, they can be carried out according to the steps and condition parameters of the existing tissue culture method of Fritillaria cirrhosa or directly using existing equipment. For example, in low-temperature plasma treatment, the discharge mode can be dielectric barrier discharge, the discharge frequency can be 5-20 kHz (such as 15 Hz), the discharge voltage can be 15-30 kV (such as 20 kV), and other parameters can be set with reference to the conventional parameters of existing low-temperature plasma treatment. The present invention document will not elaborate one by one.

[0026] The present invention will be further illustrated below with specific examples. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions indicated in the following examples are usually determined according to national standards. If there are no corresponding national standards, they are carried out according to general international standards, conventional conditions, or the conditions recommended by the manufacturer.

[0027] Example 1 In this example, the inhibitory effect of cis-3-hexen-1-ol on polyphenol oxidase was determined, including the following steps: 1. Reagent preparation Sample solutions of cis-3-hexen-1-ol inhibitors at different concentrations: Diluted to concentrations of 5 mg / L, 10 mg / L, 15 mg / L, and 30 mg / L with phosphate buffer solution (0.10 mol / L, pH = 6.8); Polyphenol oxidase solution: Prepare a 200 U / mL polyphenol oxidase solution with phosphate buffer solution (0.10 mol / L, pH = 6.8) and store it in an ice bath.

[0028] Levodopa solution: Prepare 5 mmol / L Levodopa solution with phosphate buffer (0.10 mol / L, pH = 6.8); Stop solution: 2% HCl solution.

[0029] 2. Add sample In a 96-well ELISA plate, 50 μL of different concentrations of cis-3-hexene-1-ol inhibitor sample solution and 50 μL of 200 U / mL polyphenol oxidase solution were added to each well as sample groups; at the same time, a positive control group was set up: phosphate buffer (50 μL) + polyphenol oxidase solution (50 μL); a negative control group was set up: phosphate buffer (100 μL); and an inhibitor solution blank group was set up: cis-3-hexene-1-ol inhibitor sample solution (50 μL) + phosphate buffer (50 μL). Three parallel wells were set up for each sample.

[0030] 3. Oscillation combination The ELISA plate was placed on a shaker and shaken at 300 rpm for 5 min to allow cis-3-hexen-1-ol and polyphenol oxidase to fully combine.

[0031] 4. Incubate at 25°C Place the ELISA plate in an aluminum lunch box and incubate it in a 25°C water bath shaker (100 rpm) for 10 min.

[0032] 5. Add levodopa solution Add 50 μL of 5 mmol / L L-DOPA solution to each well.

[0033] 6. Incubate at 37°C The ELISA plate was placed in a 37°C water bath shaker (100 rpm) and incubated for 10 min.

[0034] 7. OD value determination Add the stop solution, take out the ELISA plate, wipe the bottom with filter paper, and quickly place it in the ELISA instrument. Use phosphate buffer solution (0.10 mol / L, pH = 6.8) as the blank control and measure the OD value at 475 nm.

[0035] 8. Calculation of inhibition rate Inhibition rate (%) = [1 - (OD of sample group - OD of inhibitor solution blank group) / (OD of positive control group - OD of negative control group)] × 100%, calculate the average inhibition rate of each sample.

[0036] The test results showed that when the working concentration of cis-3-hexen-1-ol was 5 - 30 mg / L, the inhibition rate (%) of polyphenol oxidase remained above 70.4%; and when the working concentration was 15 mg / L, the inhibition rate (%) of polyphenol oxidase was 92.7% ± 1.1%, and when the working concentration was 30 mg / L, the inhibition rate (%) of polyphenol oxidase > 99%. In addition, in this example, cis-3-hexenal with a concentration of 15 mg / L was used as the inhibitor sample solution for testing, and it was found that its inhibitory effect on polyphenol oxidase was not significant.

[0037] Example 2 On the basis of Example 1, in this example, cis-3-hexen-1-ol was further applied to the tissue culture method of Fritillaria cirrhosa, including the following steps: Selecting fresh and healthy Fritillaria cirrhosa bulbs in March as explants, after cleaning them thoroughly, performing pre-inoculation pretreatment, pre-culture, induction culture, subculture, proliferation culture and rooting culture in sequence; Among them, the pre-inoculation pretreatment includes the following process: Performing low-temperature plasma treatment on the Fritillaria cirrhosa bulbs, using a mixed gas composed of argon and oxygen with a volume ratio of 7:1 and a flow rate of 120 ml / min as the working medium, with a discharge power of 26 W and a treatment time of 40 seconds; then cutting them into cubes with a particle size of 0.5 - 0.8 cm and soaking them in an aqueous solution containing 2.0 g / L polyvinylpyrrolidone and 4 g / L carboxymethyl chitosan for 15 min; The working condition parameters of the pre-culture include: Inoculating on the following medium: MS with a pH of 5.8 + 30 g / L sucrose + 6.2 g / L agar + 0.8 mg / L 6-BA + 1.0 mg / L NAA + 0.4 mg / L 2,4-D + 12 mg / L cis-3-hexen-1-ol; Culturing at a temperature of 22 °C, an air relative humidity of 75%, under light for 8 days, with a light time of 12 hours / day and a light intensity of 800 Lux; The working condition parameters of the induction culture include: Inoculating on the following medium: MS with a pH of 5.8 + 30 g / L sucrose + 6.8 g / L agar + 0.8 mg / L IBA + 1.2 mg / L NAA + 0.6 mg / L 2,4-D + 16 mg / L cis-3-hexen-1-ol; Culturing at a temperature of 22 °C, an air relative humidity of 75%, with a light time of 12 hours / day, a light intensity of 2000 Lux, and a culture time of 13 days; The working condition parameters for subculture include: inoculation on the following medium: MS + 30 g / L sucrose + 6.8 g / L agar + 0.8 mg / L IBA + 1.2 mg / L KT + 0.2 g / L activated carbon with a pH of 5.8; culture at a temperature of 22 °C, relative air humidity of 75%, light time of 12 hours / day, light intensity of 2000 Lux, and culture time of 16 days; The working condition parameters for proliferation culture include: inoculation on the following medium: MS + 30 g / L sucrose + 6.8 g / L agar + 0.8 mg / L IBA + 1.2 mg / L NAA + 0.8 mg / L 2,4-D with a pH of 5.8; culture at a temperature of 22 °C, relative air humidity of 75%, light time of 12 hours / day, light intensity of 1700 Lux, and culture time of 17 days; The working condition parameters for rooting culture include: inoculation on the following medium: MS + 30 g / L sucrose + 6.8 g / L agar + 0.8 mg / L IBA + 1.8 mg / L KT + 1.6 mg / L NAA with a pH of 5.8; culture at a temperature of 22 °C, relative air humidity of 75%, light time of 12 hours / day, light intensity of 2500 Lux, and acclimatization and transplantation are carried out when the small bulbs of Fritillaria cirrhosa grow to 5 - 6 cm in height and 5 - 10 roots grow out.

[0038] According to the formula: Browning rate (%) = Number of browning plants / Total number of inoculated plants × 100%; Induction rate (%) = Number of effective plants (not contaminated and not browned) / Total number of inoculated plants × 100%, the browning rate in the tissue culture method of Fritillaria cirrhosa in this example is 9.8% and the induction rate is 71.3%.

[0039] Example 3 On the basis of Example 1, in this example, cis-3-hexen-1-ol is further applied to the tissue culture method of Fritillaria cirrhosa, including the following steps: Select fresh and healthy Fritillaria cirrhosa bulbs in March as explants, and after cleaning, carry out pre-inoculation pretreatment, pre-culture, induction culture, subculture, proliferation culture and rooting culture in sequence; Among them, the pre-inoculation pretreatment includes the following process: Carry out low-temperature plasma treatment on the Fritillaria cirrhosa bulbs, use a mixed gas composed of argon and oxygen with a volume ratio of 5:1 at a flow rate of 100 ml / min as the working medium, with a discharge power of 20 W and a treatment time of 50 seconds; then cut into blocks with a particle size of 0.5 - 0.8 cm and soak in an aqueous solution containing 1.5 g / L polyvinylpyrrolidone and 3 g / L carboxymethyl chitosan for 15 min; The working condition parameters of the pre-culture include: inoculation on the following medium: MS with a pH of 5.8 + 25 g / L sucrose + 5.5 g / L agar + 0.5 mg / L 6-BA + 0.8 mg / L NAA + 0.2 mg / L 2,4-D + 10 mg / L cis-3-hexen-1-ol; the culture conditions are a temperature of 22 °C, an air relative humidity of 75%, light culture for 10 days, a light duration of 12 hours per day, and a light intensity of 700 Lux; The working condition parameters of the induction culture include: inoculation on the following medium: MS with a pH of 5.8 + 25 g / L sucrose + 6.5 g / L agar + 0.5 mg / L IBA + 0.8 mg / L NAA + 0.4 mg / L 2,4-D + 15 mg / L cis-3-hexen-1-ol; the culture conditions are a temperature of 22 °C, an air relative humidity of 75%, a light duration of 12 hours per day, a light intensity of 1950 Lux, and a culture time of 15 days; The working condition parameters of the subculture include: inoculation on the following medium: MS with a pH of 5.8 + 25 g / L sucrose + 6.5 g / L agar + 0.5 mg / L IBA + 1.0 mg / L KT + 0.1 g / L activated carbon; the culture conditions are a temperature of 22 °C, an air relative humidity of 75%, a light duration of 12 hours per day, a light intensity of 1900 Lux, and a culture time of 17 days; The working condition parameters of the proliferation culture include: inoculation on the following medium: MS with a pH of 5.8 + 25 g / L sucrose + 6.5 g / L agar + 0.5 mg / L IBA + 0.8 mg / L NAA + 0.5 mg / L 2,4-D; the culture conditions are a temperature of 22 °C, an air relative humidity of 75%, a light duration of 12 hours per day, a light intensity of 1600 Lux, and a culture time of 18 days; The working condition parameters of the rooting culture include: inoculation on the following medium: MS with a pH of 5.8 + 25 g / L sucrose + 6.5 g / L agar + 0.5 mg / L IBA + 1.5 mg / L KT + 1.5 mg / L NAA; the culture conditions are a temperature of 22 °C, an air relative humidity of 75%, a light duration of 12 hours per day, a light intensity of 2400 Lux. When the small bulbs of Fritillaria cirrhosa grow to 5 - 6 cm in height and have 5 - 10 roots, acclimatization and transplanting are carried out.

[0040] According to the formula: Browning rate (%) = Number of browning plants / Total number of inoculated plants × 100%; Induction rate (%) = Number of effective plants (not contaminated and not browned) / Total number of inoculated plants × 100%, the browning rate in the tissue culture method of Fritillaria cirrhosa in this example is 17.1% and the induction rate is 59.7%.

[0041] Example 4 On the basis of Example 1, in this example, cis-3-hexen-1-ol is further applied to the tissue culture method of Fritillaria cirrhosa, including the following steps: Select the fresh and healthy bulbs of Fritillaria cirrhosa in March as explants. After cleaning them, perform pre-inoculation pretreatment, pre-culture, induction culture, subculture, proliferation culture, and rooting culture in sequence; Among them, the pre-inoculation pretreatment includes the following process: Perform low-temperature plasma treatment on the bulbs of Fritillaria cirrhosa, use a mixed gas composed of argon and oxygen with a volume ratio of 8:1 and a flow rate of 150 ml / min as the working medium, with a discharge power of 30 W and a treatment time of 30 seconds; then cut them into blocks with a particle size of 0.5 - 0.8 cm and soak them in an aqueous solution containing 2.5 g / L polyvinylpyrrolidone and 4 g / L carboxymethyl chitosan for 15 min; The working condition parameters of the pre-culture include: Inoculate on the following medium: MS with a pH of 5.8 + 35 g / L sucrose + 6.5 g / L agar + 1.0 mg / L 6 - BA + 1.5 mg / L NAA + 0.6 mg / L 2,4 - D + 14 mg / L cis-3-hexen-1-ol; Culture at a temperature of 22°C, an air relative humidity of 75%, under light for 7 days, with a light time of 12 hours / day and a light intensity of 900 Lux; The working condition parameters of the induction culture include: Inoculate on the following medium: MS with a pH of 5.8 + 30 g / L sucrose + 7.0 g / L agar + 1.0 mg / L IBA + 1.5 mg / L NAA + 0.8 mg / L 2,4 - D + 18 mg / L cis-3-hexen-1-ol; Culture at a temperature of 22°C, an air relative humidity of 75%, with a light time of 12 hours / day, a light intensity of 2050 Lux, and a culture time of 12 days; The working condition parameters of the subculture include: Inoculate on the following medium: MS with a pH of 5.8 + 30 g / L sucrose + 7.0 g / L agar + 1.0 mg / L IBA + 1.5 mg / L KT + 0.3 g / L activated carbon; Culture at a temperature of 22°C, an air relative humidity of 75%, with a light time of 12 hours / day, a light intensity of 2050 Lux, and a culture time of 14 days; The working condition parameters of the proliferation culture include: Inoculate on the following medium: MS with a pH of 5.8 + 30 g / L sucrose + 7.0 g / L agar + 1.0 mg / L IBA + 1.5 mg / L NAA + 1.0 mg / L 2,4 - D; Culture at a temperature of 22°C, an air relative humidity of 75%, with a light time of 12 hours / day, a light intensity of 1800 Lux, and a culture time of 15 days; The working condition parameters for rooting culture include: inoculation on the following medium: MS + 30 g / L sucrose + 7.0 g / L agar + 1.0 mg / L IBA + 2.0 mg / L KT + 2.0 mg / L NAA with a pH of 5.8; culturing at a temperature of 22°C, an air relative humidity of 75%, a light duration of 12 hours per day, and a light intensity of 2600 Lux. When the small bulbs of Fritillaria cirrhosa grow to 5 - 6 cm in height and 5 - 10 roots emerge, acclimatization and transplantation are carried out.

[0042] According to the formulas: Browning rate (%) = Number of browning plants / Total number of inoculated plants × 100%; Induction rate (%) = Number of effective plants (non - contaminated and non - browned) / Total number of inoculated plants × 100%, it is calculated that the browning rate in the tissue culture method of Fritillaria cirrhosa in this example is 14.3% and the induction rate is 63.6%.

[0043] Comparative Example 1 This example provides a tissue culture method for Fritillaria cirrhosa, which is only different from Example 2 in that: 1) The low - temperature plasma treatment in the pre - inoculation pretreatment is adjusted to a traditional disinfection treatment (soaking in 75% ethanol for 10 s, rinsing twice with sterile water, soaking in 0.1% mercuric chloride solution for 7 min, and rinsing twice with sterile water, 2 minutes each time); 2) cis - 3 - hexen - 1 - ol is not added to the medium used during pre - culture and induction culture; the remaining steps and parameters are the same.

[0044] According to the formulas: Browning rate (%) = Number of browning plants / Total number of inoculated plants × 100%; Induction rate (%) = Number of effective plants (non - contaminated and non - browned) / Total number of inoculated plants × 100%, it is calculated that the browning rate in the tissue culture method of Fritillaria cirrhosa in this example is 69.1% and the induction rate is 16.5%.

[0045] Comparative Example 2 This example provides a tissue culture method for Fritillaria cirrhosa, which is only different from Example 2 in that: The low - temperature plasma treatment in the pre - inoculation pretreatment is adjusted to a traditional disinfection treatment (soaking in 75% ethanol for 10 s, rinsing twice with sterile water, soaking in 0.1% mercuric chloride solution for 7 min, and rinsing twice with sterile water, 2 minutes each time); the remaining steps and parameters are the same.

[0046] According to the formulas: Browning rate (%) = Number of browning plants / Total number of inoculated plants × 100%; Induction rate (%) = Number of effective plants (non - contaminated and non - browned) / Total number of inoculated plants × 100%, it is calculated that the browning rate in the tissue culture method of Fritillaria cirrhosa in this example is 57.8% and the induction rate is 29.1%.

[0047] Comparative Example 3 This example provides a tissue culture method for Fritillaria cirrhosa, which is only different from Example 2 in that: cis-3-hexen-1-ol is not added to the culture medium used during pre-culture and induction culture; the remaining steps and parameters are the same.

[0048] According to the formula: Browning rate (%) = Number of browning plants / Total number of inoculated plants × 100%; Induction rate (%) = Number of effective plants (not contaminated and not browned) / Total number of inoculated plants × 100%, it is calculated that the browning rate in the tissue culture method of Fritillaria cirrhosa in this example is 42.4%, and the induction rate is 38.7%.

[0049] Comparative Example 4 This example provides a tissue culture method for Fritillaria cirrhosa, which is only different from Example 2 in that: during the pre-treatment before inoculation, the discharge power in the low-temperature plasma treatment is adjusted to 150 W, and the treatment time is adjusted to 2 minutes; the remaining steps and parameters are the same.

[0050] According to the formula: Browning rate (%) = Number of browning plants / Total number of inoculated plants × 100%; Induction rate (%) = Number of effective plants (not contaminated and not browned) / Total number of inoculated plants × 100%, it is calculated that the browning rate in the tissue culture method of Fritillaria cirrhosa in this example is 54.6%, and the induction rate is 33.8%.

[0051] In summary, the embodiments of the present invention provide a tissue culture method for Fritillaria cirrhosa. Based on the existing tissue culture method for Fritillaria cirrhosa, the present invention proposes a new tissue culture method for Fritillaria cirrhosa based on the synergistic effect of low-temperature plasma pre-treatment and chemical induction with cis-3-hexen-1-ol, which can effectively inhibit the browning phenomenon during the culture process, improve the culture efficiency, and has the characteristics of a low browning rate and a high induction rate, providing a new means for the rapid large-scale propagation of Fritillaria cirrhosa.

[0052] The various embodiments of the present invention may exist in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity, and should not be construed as a rigid limitation on the scope of the present invention; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single values within that range. For example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and single numbers within that range, such as 1, 2, 3, 4, 5, and 6, regardless of the range. Additionally, whenever a numerical range is indicated herein, it means including any cited number (fraction or integer) within the indicated range.

[0053] The above are only specific embodiments of the present invention, enabling those skilled in the art to understand or implement the present invention. Various modifications to these embodiments will be obvious to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein, but rather to the broadest scope consistent with the principles and novel features claimed herein.

Claims

1. A tissue culture method for Fritillaria cirrhosa, characterized in that: The tissue culture method of Fritillaria cirrhosa comprises: selecting fresh and healthy Fritillaria cirrhosa bulbs as explants, and then sequentially performing pre-inoculation pretreatment, pre-culture, induction culture, subculture, proliferation culture and rooting culture; The pretreatment before inoculation includes the following process: subjecting the bulbs of Fritillaria cirrhosa to low-temperature plasma treatment, then cutting and soaking them in an aqueous solution containing polyvinyl pyrrolidone and carboxymethyl chitosan; The working condition parameters of the low-temperature plasma treatment include: a mixed gas consisting of argon and oxygen with a flow rate of 100-150 ml / min as the working medium, a discharge power of 20-30 W, and a treatment time of 30-50 seconds; The culture medium used in the pre-culture and the induction culture both contains cis-3-hexen-1-ol.

2. The tissue culture method of Fritillaria cirrhosae according to claim 1, characterized in that: The volume ratio of the argon gas to the oxygen gas is (5-8):1, the discharge power is 26W, and the processing time is 40 seconds.

3. The tissue culture method of Fritillaria cirrhosae according to claim 1, characterized in that: The concentration of the polyvinyl pyrrolidone is 1.5-2.5 g / L.

4. The tissue culture method of Fritillaria cirrhosae according to claim 1, characterized in that: The concentration of the carboxymethyl chitosan is 3-5 g / L.

5. The tissue culture method of Fritillaria cirrhosae according to claim 1, characterized in that: The working condition parameters of the pre-culture include: Inoculate in the following medium: MS with a pH of 5.5-6.0 + 25-35 g / L sucrose + 5.5-6.5 g / L agar + 0.5-1.0 mg / L 6-BA + 0.8-1.5 mg / L NAA + 0.2-0.6 mg / L 2,4-D + 10-14 mg / L cis-3-hexen-1-ol; The culture temperature is 21~23℃, the relative humidity of the air is 70~80%, the light culture is 7~10 days, the light time is 12 hours / day, and the light intensity is 700~900Lux.

6. The tissue culture method of Fritillaria cirrhosae according to claim 1, characterized in that: The working condition parameters of the induction culture include: Inoculate in the following medium: MS with a pH of 5.5-6.0 + 25-35 g / L sucrose + 6.5-7.0 g / L agar + 0.5-1.0 mg / L IBA + 0.8-1.5 mg / L NAA + 0.4-0.8 mg / L 2,4-D + 15-18 mg / L cis-3-hexen-1-ol; The culture temperature is 21~23℃, the relative humidity of the air is 70~80%, the light time is 12 hours / day, the light intensity is 1950~2050Lux, and the culture time is 12~15 days.

7. The tissue culture method of Fritillaria cirrhosae according to claim 1, characterized in that: The working condition parameters of the subculture include: Inoculate in the following medium: MS with a pH of 5.5-6.0 + 25-35 g / L sucrose + 6.5-7.0 g / L agar + 0.5-1.0 mg / L IBA + 1.0-1.5 mg / L KT + 0.1-0.3 g / L activated carbon; The culture temperature is 21~23℃, the relative humidity of the air is 70~80%, the light time is 12 hours / day, the light intensity is 1950~2050Lux, and the culture time is 14~17 days.

8. The tissue culture method of Fritillaria cirrhosae according to claim 1, characterized in that: The working condition parameters of the proliferation culture include: Inoculate in the following medium: MS with a pH of 5.5-6.0 + 25-35 g / L sucrose + 6.5-7.0 g / L agar + 0.5-1.0 mg / L IBA + 0.8-1.5 mg / L NAA + 0.5-1.0 mg / L 2,4-D; The culture temperature is 21~23℃, the relative humidity is 70~80%, the light time is 12 hours / day, the light intensity is 1600~1800Lux, and the culture time is 15~18 days.

9. The tissue culture method of Fritillaria cirrhosae according to claim 1, characterized in that: The working condition parameters of the rooting culture include: Inoculate in the following medium: MS with a pH of 5.5-6.0 + 25-35 g / L sucrose + 6.5-7.0 g / L agar + 0.5-1.0 mg / L IBA + 1.5-2.0 mg / L KT + 1.5-2.0 mg / L NAA; The cultivation temperature is 21~23℃, the relative humidity of the air is 70~80%, the light time is 12 hours / day, and the light intensity is 2400~2600Lux. When the bulblets of Fritillaria cirrhosa grow to 5~6cm in height and 5-10 roots are grown, the seedlings are hardened and transplanted.